Functional identity of receptors for proteolysis-inducing factor on human and murine skeletal muscle.

Mirza KA, Tisdale MJ - Br. J. Cancer (2014)

Bottom Line:
Both murine and human PIF inhibited total protein synthesis and stimulated protein degradation in human and murine myotubes to about the same extent, and this was attenuated by a rabbit polyclonal antibody to the murine PIF receptor, but not by a non-specific rabbit antibody.Both murine and human PIF increased the activity of the ubiquitin-proteasome pathway in both human and murine myotubes, as evidenced by an increased 'chymotrypsin-like' enzyme activity, protein expression of the 20S and 19S proteasome subunits, and increased expression of the ubiquitin ligases MuRF1 and MAFbx, and this was also attenuated by the anti-mouse PIF receptor antibody.These results suggest that the murine and human PIF receptors are identical.

Background: Cachexia in both mice and humans is associated with tumour production of a sulphated glycoprotein called proteolysis-inducing factor (PIF). In mice PIF binds with high affinity to a surface receptor in skeletal muscle, but little is known about the human receptor. This study compares the human PIF receptor with the murine.

Methods: Human PIF was isolated from the G361 melanoma and murine PIF from the MAC16 colon adenocarcinoma. The human PIF receptor was isolated from human skeletal muscle myotubes. Protein synthesis and degradation induced by human and murine PIF was studied in human and murine skeletal muscle myotubes.

Results: Both the human and murine PIF receptors showed the same immunoreactivity and Mr 40 000. Both murine and human PIF inhibited total protein synthesis and stimulated protein degradation in human and murine myotubes to about the same extent, and this was attenuated by a rabbit polyclonal antibody to the murine PIF receptor, but not by a non-specific rabbit antibody. Both murine and human PIF increased the activity of the ubiquitin-proteasome pathway in both human and murine myotubes, as evidenced by an increased 'chymotrypsin-like' enzyme activity, protein expression of the 20S and 19S proteasome subunits, and increased expression of the ubiquitin ligases MuRF1 and MAFbx, and this was also attenuated by the anti-mouse PIF receptor antibody.

Conclusions: These results suggest that the murine and human PIF receptors are identical.

fig3: Effect of anti-PIF-receptor antibody on PIF-induced protein degradation in murine myotubes (A) and (B) and human myotubes (C) and (D). Total protein degradation in C2C12 myotubes after 24 h incubation with either murine (A) or human (B) PIF (4.2 nM) and in human myotubes incubated with either murine (C) or human (D) PIF (4.2 nM) in the absence or presence of rabbit anti-murine PIFR antibody (rAb; 5 μg ml−1), or non-specific rabbit antibody (nsAb; 5 μg ml−1). Both antibodies were added 2 h prior to PIF. The experiment was repeated three times. Differences from control are indicated as b, P<0.01 or c, P<0.001, while differences in the presence of antibody are shown as e, P<0.01 or f, P<0.001.

Mentions:
In murine myotubes both human and murine PIF also significantly stimulated total protein degradation, as determined by the release of [3H] phenylalanine from pre-labelled cells (Figure 3A and B), and in both cases this was attenuated by a polyclonal antibody to the mPIFR, but not by a non-specific polyclonal antibody. The extent of protein degradation by human and murine PIF was approximately the same. In human myotubes both murine and human PIF also initiated an increase in total protein degradation (Figure 3C and D), to about the same extent. Again the protein degradation induced by both human and murine PIF was completely attenuated by the rabbit polyclonal antibody to the mPIFR, but not by a non-specific rabbit polyclonal antibody (Figure 3C and D).

fig3: Effect of anti-PIF-receptor antibody on PIF-induced protein degradation in murine myotubes (A) and (B) and human myotubes (C) and (D). Total protein degradation in C2C12 myotubes after 24 h incubation with either murine (A) or human (B) PIF (4.2 nM) and in human myotubes incubated with either murine (C) or human (D) PIF (4.2 nM) in the absence or presence of rabbit anti-murine PIFR antibody (rAb; 5 μg ml−1), or non-specific rabbit antibody (nsAb; 5 μg ml−1). Both antibodies were added 2 h prior to PIF. The experiment was repeated three times. Differences from control are indicated as b, P<0.01 or c, P<0.001, while differences in the presence of antibody are shown as e, P<0.01 or f, P<0.001.

Mentions:
In murine myotubes both human and murine PIF also significantly stimulated total protein degradation, as determined by the release of [3H] phenylalanine from pre-labelled cells (Figure 3A and B), and in both cases this was attenuated by a polyclonal antibody to the mPIFR, but not by a non-specific polyclonal antibody. The extent of protein degradation by human and murine PIF was approximately the same. In human myotubes both murine and human PIF also initiated an increase in total protein degradation (Figure 3C and D), to about the same extent. Again the protein degradation induced by both human and murine PIF was completely attenuated by the rabbit polyclonal antibody to the mPIFR, but not by a non-specific rabbit polyclonal antibody (Figure 3C and D).

Bottom Line:
Both murine and human PIF inhibited total protein synthesis and stimulated protein degradation in human and murine myotubes to about the same extent, and this was attenuated by a rabbit polyclonal antibody to the murine PIF receptor, but not by a non-specific rabbit antibody.Both murine and human PIF increased the activity of the ubiquitin-proteasome pathway in both human and murine myotubes, as evidenced by an increased 'chymotrypsin-like' enzyme activity, protein expression of the 20S and 19S proteasome subunits, and increased expression of the ubiquitin ligases MuRF1 and MAFbx, and this was also attenuated by the anti-mouse PIF receptor antibody.These results suggest that the murine and human PIF receptors are identical.

Background: Cachexia in both mice and humans is associated with tumour production of a sulphated glycoprotein called proteolysis-inducing factor (PIF). In mice PIF binds with high affinity to a surface receptor in skeletal muscle, but little is known about the human receptor. This study compares the human PIF receptor with the murine.

Methods: Human PIF was isolated from the G361 melanoma and murine PIF from the MAC16 colon adenocarcinoma. The human PIF receptor was isolated from human skeletal muscle myotubes. Protein synthesis and degradation induced by human and murine PIF was studied in human and murine skeletal muscle myotubes.

Results: Both the human and murine PIF receptors showed the same immunoreactivity and Mr 40 000. Both murine and human PIF inhibited total protein synthesis and stimulated protein degradation in human and murine myotubes to about the same extent, and this was attenuated by a rabbit polyclonal antibody to the murine PIF receptor, but not by a non-specific rabbit antibody. Both murine and human PIF increased the activity of the ubiquitin-proteasome pathway in both human and murine myotubes, as evidenced by an increased 'chymotrypsin-like' enzyme activity, protein expression of the 20S and 19S proteasome subunits, and increased expression of the ubiquitin ligases MuRF1 and MAFbx, and this was also attenuated by the anti-mouse PIF receptor antibody.

Conclusions: These results suggest that the murine and human PIF receptors are identical.